ABSTRACT Liganded estrogen receptor (ER) bound to target gene enhancers activates transcription through sequential interactions with a number of coactivators. These coactivators act first to make local chromatin sites more accessible and, subsequently, in conjunction with the Mediator, establish functional Pol II preinitiation complexes (PICs) at core promoters. We have shown both a critical requirement for MED1, an NR box-containing Mediator subunit, in normal mammary gland development and in mammary tumor formation. Aberrant interactions of tamoxifen-liganded ER with MED1 have also been implicated in acquisition of resistance to tamoxifen therapy. Other coactivators that display interactions and/or cooperative functions with Mediator include SRCs, PGC1, CCAR1, and CoCoA. We also have evidence for direct ER interactions with the MLL3/4 complex, which possesses histone H3K4 monomethyltransferase and H3K27 demethylase activities and, along with pioneer factor FOXA1, functions at the earliest stages of enhancer activation. Partly in light of an emerging role of Mediator in enhancer-promoter communication, we hypothesize (i) that various of these cofactors facilitate ER- MED1/Mediator interactions and functions by acting as intermediary factors in the transitions from enhancer activation and chromatin remodeling/modification to PIC formation and function and (ii) that these interactions go awry in tamoxifen resistance to facilitate transcription under otherwise repressive conditions. Here, we propose to elucidate mechanistic details of key ER-cofactor interactions through stages of enhancer activation, enhancer-promoter interactions and PIC assembly/function. In Aim 1, we will use integrated biochemical (in vitro transcription), cell-based (genome-wide analyses in combination with CRISPR/Cas9-mediated mutant MED1 knockins) and mouse tumor xenograft approaches to elucidate (i) the role and mechanism of MED1 NR box- dependent recruitment/function of Mediator by ER; (ii) the role and mechanism of an alternative, MED1 NR box- independent pathway for Mediator recruitment by ER; and (iii) the mechanism by which crosstalk between the tyrosine kinase HER2 and the (phosphorylated) MED1 subunit of Mediator contributes to resistance to tamoxifen therapy. In Aim 2, we will employ similar approaches to investigate (i) establishment of active enhancer landscapes bearing H3K4me1 and H3K27ac marks, with emphasis on mechanisms by which these modifications are effected through ER- and FOXA1-based cooperativity between p300/CBP, MLL3/4C, and SRC and PGC-1b coactivators; and (ii) distal enhancer function through promoter interactions, with emphasis on the in vitro recapitulation and mechanistic analysis of Mediator-dependent ER function through additional factors (including cohesin). In Aim 3, we will employ cryo-EM, XL-MS and computational integrative modeling to elucidate details of physical interactions between ER and critical cofactors (Mediator and MLL3/4C). Based on this, we will design and test stabilized peptidomimetic and related therapeutic agents that target key protein-protein interactions. Thus, our studies will have widespread impact from both nuclear receptor and breast cancer perspectives.